. The goal of these studies is an increased understanding of the specificity determinants for the interaction of thrombin with its substrates, inhibitors, and other ligands. This information is expected to contribute to an increased understanding of the pathogenesis of thrombotic disorders and thereby assist in development of pharmacologic agents. A principal tool in this investigation will be the dysthrombin, Thrombin Quick I. This work, extending previous findings, will include further study of the thrombin-fibrinogen interaction and an examination of reactions of thrombin which have not previously been investigated with Thrombin Quick I. To further assess specificity determinants in the release of fibrinopeptide A the activity of thrombin and Thrombin Quick I acting on the polypeptides from the N-DSK fragment of fibrinogen will be compared. Determination of the kinetic parameters for the release of fibrinopeptide B from fibrin I (fibrinogen from which fibrinopeptide A has been removed) will be completed and the results compared to those obtained previously for fibrinopeptide A. Other reactions to be investigated include inhibition by heparin cofactor II, the heparin enhancement of antithrombin III inhibition. Factor XIII activation, the hydrolysis of Protein S, binding to hirudin, binding (inhibition by) platelet protease nexin, and regulation of calcium fluxes in platelet stimulation. The nature of the defect in catalysis by Thrombin Quick I will be further investigated by looking for a conformational change on binding fibrinogen, and examining the relative reactivity of histidine at the catalytic site. To complement these studies, a congenital dysfibrinogen, Fibrinogen Baton Rouge will be characterized with respect to both its structural and functional defects. The final phase of these studies will be work with site directed mutants of thrombin to assess residues critical in the thrombin-fibrinogen interaction. These studies will focus on modification of residues 385, 388, 391, 476 in the human prothrombin sequence which are located within the major fibrinogen binding groove on the thrombin surface, as recently demonstrated by the crystal structure. The role of each of these residues in the thrombin-fibrinogen interaction will be assessed by comparing the kinetic constants for fibrinopeptide A release to those obtained for r-thrombin.